The present invention relates to a polypeptide of glycosaminoglycan sulfotransferase originating from human and a DNA coding for the polypeptide.
Chondroitin sulfate is a representative sulfated mucopolysaccharide (glycosaminoglycan). Chondroitin sulfate proteoglycan (CSPG) exists abundantly in cartilage, and is considered to participate in expression and maintenance of the phenotype of chondrocyte (Tsukahara, T., Okamura, M., Suzuki, S., Iwata, H., Miura, T., and Kimata, K. (1991) J. Cell Sci. 100, 387-395). CSPG exists also in various tissues other than cartilage, and is considered to play an important role for intercellular interactions (Kjellen, L. and Lindahl, U. (1991) Annu. Rev. Biochem. 60, 443-475).
Major chondroitin sulfate found in mammalian and avian tissues has sulfate groups at C-6 or C-4 positions of acetylgalactosamine residues. The following knowledge has been obtained for the ratio of 6-sulfation/4-sulfation. (i) The ratio of chondroitin 6-sulfate/chondroitin 4-sulfate (6/4 ratio) increases along with progress of final differentiation of cartilage. (ii) The 6/4 ratio in CSPG decreases in skin of rat along with passage of days after the birth. (iii) When CSPG and dermatan sulfate proteoglycan (DSPG) present in arterial smooth muscle cells are compared between an atherosclerosis-resistant pigeon and an atherosclerosis-sensitive pigeon, the major component is chondroitin 4-sulfate in the former species, whereas the major component is chondroitin 6-sulfate in the latter species. (iv) When monocytic leukemia cells (Ml) are cultured under the culture condition successively changed to suit for cell proliferation, inhibition of high-density proliferation and induction of differentiation to macrophage, the 6/4 ratio in CSPG is decreasing as the condition is changed in the above-defined order, and almost only chondroitin 4-sulfate is produced under the final differentiation-inducing state. (v) As a result of comparison of human colon normal tissue with human colon tumor tissue, chondroitin 6-sulfate and chondroitin in PG are increased in the tumor tissue. (vi) When mouse osteoblast cells are compared before and after calcification, the 6/4 ratio of DSPG is decreased in the cells after calcification. (vii) When platelet derived growth factor (PDGF) is added to a culture medium of monkey arterial smooth muscle cells, the 6/4 ratio of versican-like CSPG is increased as compared with a control with no addition of PDGF (Glycobiology Series (I), "Diversified World of Saccharides", Kodansha, pp. 164, 166).
It has been also reported for chondroitin sulfate that two sulfate groups exist per repeating disaccharide unit. For example, GlcA.beta.1.fwdarw.3GalNAc(4,6-bisS) has been found in: subcultured chick embryo chondrocytes; mouse mast cells differentiated from myeloid cells cultivated in a medium supplemented with a conditioned medium derived from mouse spleen cell cultures; rat glomeruli; culture liquid of organotypic cultured human colonic mucosa; rat serosa mast cells; secretory granules of human lung mast cells; human monocytes activated by phorbol myristate acetate and macrophages derived from such monocytes; mouse osteoblasts; rat glomerulus vascular membrane cells; and sea cucumber parietal cells. In addition, GalNAc(4,6-bisS) has been found in the nonreducing terminal of the chondroitin sulfate from chick embryo epiphyseal cartilage, rat processus xiphoideus cartilage, and cell layer obtained by cultivating chick embryo chondrocyte. Further, GalNAc(4,6-bisS).beta.1.fwdarw.4GlcA.beta.1.fwdarw.3GalNAc(4,6-bisS) has been found in the nonreducing terminal of the chondroitin sulfate from thrombomodulin extracted and purified from rabbit lung. Moreover, GlcA(2S)-GalNAc(6S) has been found in the nonreducing terminal of the chondroitin sulfate from mast cells originating from mouse lymph node (Glycobiology Series (I), "Diversified World of Saccharides," Kodansha, p.166).
It is considered that the diversification of the sulfation pattern of chondroitin sulfate as described above reflects a molecular basis of the function of chondroitin sulfate. It is also considered that sulfation plays an important role in expression of physiological activities of chondroitin sulfate. Considering the importance of sulfation in expression of physiological activities of chondroitin sulfate, it is expected that a method for sulfating a specific site of chondroitin sulfate is indispensable to analyze physiological activities of chondroitin sulfate and modify its function. Sulfation at a specific site of a sugar residue of glycosaminoglycan is catalyzed by a sulfotransferase specific to the site.
If a gene of sulfotransferase for glycosaminoglycan is cloned, information on substrate specificity concerning the acceptor may be obtained, providing an approach useful to study the relationship between structure and function of glycosaminoglycan. It is assumed that various types of glycosaminoglycan sulfotransferases participate in synthesis of glycosaminoglycan. However, cloning of cDNA of sulfotransferase is difficult. In fact, those having been cloned only include cDNA's of N-sulfotransferase/N-deacetylase from rat liver, heparin-producing cell line, and mouse mast cell tumor.
The present inventors have apparently homogenously purified, from a culture supernatant of chick chondrocytes cultured in a serum-free medium, chondroitin 6-sulfotransferase (hereinafter often abbreviated as "C6ST") which transfers sulfate group from 3'-phosphoadenosine 5'-phosphosulfate to the C-6 position of N-acetylgalactosamine residue of glycosaminoglycan such as chondroitin (Habuchi, O., Matsui, Y., Kotoya, Y., Aoyama, Y., Yasuda, Y., and Noda, M. (1993) J. Biol. Chem. 268, 21968-21974). Further, the present inventors have prepared oligonucleotide primers on the basis of the partial amino acid sequence of this enzyme, performed the chick cDNA cloning with the aid of the primers and proved that C6ST activity is expressed by the polypeptide obtained from this DNA. The present inventors have also found that this enzyme has an activity to transfer sulfate group to the C-6 position of galactose residue of keratan sulfate (Fukuta, M., Uchimura, K., Nakashima, K., Kato, M., Kimata, K., Shinomura, T. and Habuchi, O. (1995) J. Biol. Chem. 270, 18575-18580).
However, it has been yet unknown of the DNA that codes for the polypeptide of chondroitin 6-sulfotransferase originating from human the application of which to pharmaceuticals may be expected.
Considering the importance of sulfation in expression of physiological activities of chondroitin sulfate, the enzyme, which transfers sulfate group to chondroitin sulfate, is extremely important not only to perform a study on analysis of function of chondroitin sulfate but also to provide a certain type of chondroitin sulfate in order to create pharmaceuticals having physiological activities preferred for human. Moreover, if polypeptide of chondroitin 6-sulfotransferase (C6ST) originating from human and DNA coding for the polypeptide are obtained, the medical application thereof, including gene therapy, might be expected in the form of pharmaceuticals against, or diagnostics of, human diseases attributable to a low sulfation at C-6 position of N-acetylgalactosamine residue of chondroitin sulfate, a low sulfation at C-6 position of galactose residue of keratan sulfate and the like ("low sulfation" as used herein means that the sulfation degree is low).